The success or failure of radiation therapy rests, in part, on selective killing of tumor cells over normal cells. It is poorly known which molecular mechanisms are responsible for this difference. The applicants will test the overall hypothesis that adhesion to physiological extracellular matrices provides positional survival signals to epithelial normal cells that are reduced or absent in tumor cells. It is further hypothesized that tumor cells counteract loss of matrix-dependent survival signals through inappropriate activation of growth factor receptors. In support of these hypotheses, preliminary evidence revealed that certain matrix components, including the basement membrane type IV collagen, enhance radioresistance of malignant keratinocytes when the epidermal growth factor receptor (EGFR) is blocked. The overall goal of this application is to further define molecular mechanisms and pathways by which integrin engagement and EGFR activation coordinately control epithelial cell survival. This will be done using normal keratinocytes and cells representing an early stage of keratinocyte tumor progression (HaCaT). The Specific Aims of the work proposed are to (1) define matrix components and cognate matrix adhesion receptors that contribute to radiation resistance in these cells, (2) characterize coordinate control of signaling pathways (PI-3- kinase/AKT; MEK/MAPK) that contribute to cell survival by integrin engagement and EGFR activation, (3) assess functional roles of integrins and the EGFR in survival of HaCaT cells grown as tumor cell nests in organotypic epidermal reconstructs. Epidermal reconstructs represent a uniquely versatile three-dimensional tissue model to assess complex regulation of cell survival. To assess functional roles of specific surface receptors and signaling molecules in HaCaT cell survival the investigators have established a tetracycline regulatable gene expression system in these cells allowing the conditional expression of antisense and dominant negative constructs even if they are lethal in 2D cell culture. The use of these tools in organotypic reconstructs promises to provide novel information about cell survival signaling in an 'in vivo' context with clear implications for therapeutic intervention.